X inactivation is an important mechanism of dosage compensation in mammals, which silences at random one of the two X chromosomes in female cells and thus equalizes gene expression between males (XY) and females (XX). X chromosome silencing is associated with the accumulation of inactive epigenetic marks. The inactive X chromosome is highly condensed and preferentially located adjacent to specific nuclear compartments, either the nuclear membrane or the surface of the nucleolus. Despite efficient silencing some genes escape X inactivation and thus remain bi-allelically expressed in females, resulting in sexually dimorphic gene expression levels. A number of these genes escape X inactivation in multiple species, indicating that escape from silencing is an essential feature of these genes. To track allele-specific gene expression changes during cell differentiation at single-cell resolution and determine pathways that govern X-linked gene silencing and escape during development we will employ mouse models that we previously developed to measure allele-specific gene expression and chromatin structure. Our previous findings demonstrate tissue-specific differences in genes that escape X inactivation. We will now address cell-to-cell and time-related variability in X-linked gene expression. This research will help develop analytical methods to follow dynamic changes in gene expression applicable to the whole genome. To determine the 3D architecture of the inactive X chromosome and of domains of inactivation and escape we will use a novel DNase Hi-C method combined with allele-specific analyses. By this approach we have obtained the first 3D view of the whole inactive X chromosome, which reveals a surprising bipartite structure with an apparent hinge that binds CTCF on the inactive allele. These preliminary results will serve as the basis for further experiments to obtain a higher resolution view using X chromosome capture libraries. We will examine the 3D data in relation to lncRNA loci and chromatin elements such as CTCF binding sites that may govern the architecture of the silenced X chromosome and of domains of escape and inactivation. In particular, we will examine the role of specific elements in positioning the inactive X chromosome within the nucleus. This research is relevant to the understanding of sex differences and of congenital and acquired diseases associated with sex chromosome and other genomic abnormalities.